Gene therapy has been increasingly successful in treating several single-gene defects that cause blindness. In particular, multiple successful clinical trials for Leber's congenital amaurosis type 2 (LCA2) have utilized a 25 year-old viral delivery vehicle, based on adeno-associated virus (AAV) serotype 2, to deliver a functional copy of the rpe65 gene to the retinal pigment epithelium (RPE). These trials have taken landmark strides in enhancing visual function in over 30 patients, success that has established the proof of concept that if a causative gene can be identified in a group of patients, a functional replacement gene can be packaged and safely delivered with AAV. However, as the majority of mutations underlying retinal degenerative diseases have now been identified, it has become clear that almost all encode photoreceptor-specific transcripts, establishing photoreceptors as the primary target for retinal gene therapy. Furthermore, many of these mutations are autosomal dominant, such that gene replacement strategies are not suitable. To build upon the LCA2 trial successes, at least two major hurdles that impede broader application of retinal gene therapy must thus be overcome. First, vectors based on natural AAV variants require a subretinal of the vector to mediate gene delivery to photoreceptors or RPE, with accompanying retinal detachment with the creation of a bleb between the photoreceptors and underlying RPE. This procedure damages the retina, may exacerbate the retinal degeneration, and can induce reactive gliosis. In addition, subretinal injection limits the therapeutic effect to the area of th bleb, beyond which the AAV does not spread. Gene delivery from the vitreous would be considerably less traumatic and would offer the potential for pan-retinal transduction, both of which would represent significant advances. Since no natural AAV serotypes can transduce the photoreceptors from the vitreous in either murine or non-human primate (NHP) models, we developed and implemented a directed evolution approach that, as we have recently published, has yielded a novel AAV capable of photoreceptor transduction from the vitreous in the murine and to an extent in the NHP retina. We now propose to build upon this success and engineer AAV variants for optimal therapeutic gene delivery to the NHP retina. A second problem with photoreceptor gene therapy is that many retinal degenerations are autosomal dominant. While RNAi can yield a partial knockdown of pathological alleles, a full ablation of such genes would be desirable. There have been recent advances in the development of site-specific DNA nucleases that can knock out target genes, and we will build upon these advances to knock out dominant alleles that underlie retinal degeneration. We thus propose a unique blend of molecular virology, protein engineering, and a translationally important animal model to engineer enhanced genetic delivery systems and cargo for treating human retinal disease.